CD62L as target receptor for specific gene delivery into less differentiated human T lymphocytes.

Chimeric antigen receptor (CAR)-expressing T cells are a complex and heterogeneous gene therapy product with variable phenotype compositions. A higher proportion of less differentiated CAR T cells is usually associated with improved antitumoral function and persistence. We describe in this study a novel receptor-targeted lentiviral vector (LV) named 62L-LV that preferentially transduces less differentiated T cells marked by the L-selectin receptor CD62L, with transduction rates of up to 70% of CD4+ and 50% of CD8+ primary T cells. Remarkably, higher amounts of less differentiated T cells are transduced and preserved upon long-term cultivation using 62L-LV compared to VSV-LV. Interestingly, shed CD62L neither altered the binding of 62L-LV particles to T cells nor impacted their transduction. The incubation of 2 days of activated T lymphocytes with 62L-LV or VSV-LV for only 24 hours was sufficient to generate CAR T cells that controlled tumor growth in a leukemia tumor mouse model. The data proved that potent CAR T cells can be generated by short-term ex vivo exposure of primary cells to LVs. As a first vector type that preferentially transduces less differentiated T lymphocytes, 62L-LV has the potential to circumvent cumbersome selections of T cell subtypes and offers substantial shortening of the CAR T cell manufacturing process.

Dasatinib is a potent enhancer for CAR T cell generation by CD3-targeted lentiviral vectors.

CD3-targeted lentiviral vectors (CD3-LVs) mediate selective transduction of human T lymphocytes in vitro and in vivo while simultaneously activating the targeted cells. Previously, we have demonstrated that CD3-LV leads to downmodulation of the CD3:T cell receptor (TCR) complex. We therefore hypothesized that inhibition of CD3 phosphorylation by Src/Abl tyrosine kinase inhibitors such as dasatinib results in enhancement of gene delivery by T cell-targeted LVs. Indeed, dasatinib treatment of T cells prior to incubation with CD3-LV increased reporter gene delivery by 3- to 10-fold. Moreover, the presence of dasatinib enhanced selective transduction into non-activated target cells present in whole blood. When combined with delivery of the CD19-chimeric antigen receptor (CAR) gene, dasatinib increased CAR T cell numbers by close to 10-fold. Importantly, the short-term exposure of T cells to dasatinib during vector incubation did not interfere with tumor cell killing by the resulting CAR T cells and rather came along with less upregulated exhaustion markers and a more naive phenotype. Our data suggest that dasatinib prevents CD3-LV-induced phosphorylation and CD3:TCR intake, thereby increasing the amount of CD3-LV bound to the cell surface. This is the first description of dasatinib as transduction enhancer, an activity particularly relevant for CAR T cell generation with CD3-LV.

Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8.

Preclinical studies on gene delivery into mouse lymphocytes are often hampered by insufficient activity of lentiviral (LV) and adeno-associated vectors (AAVs) as well as missing tools for cell type selectivity when considering in vivo gene therapy. Here, we selected designed ankyrin repeat proteins (DARPins) binding to murine CD8. The top-performing DARPin was displayed as targeting ligand on both vector systems. When used on engineered measles virus (MV) glycoproteins, the resulting mCD8-LV transduced CD8+ mouse lymphocytes with near-absolute (>99%) selectivity. Despite its lower functional titer, mCD8-LV achieved 4-fold higher gene delivery to CD8+ cells than conventional VSV-LV when added to whole mouse blood. Addition of mCD8-LV encoding a chimeric antigen receptor (CAR) specific for mouse CD19 to splenocytes resulted in elimination of B lymphocytes and lymphoma cells. For display on AAV, the DARPin was inserted into the GH2-GH3 loop of the AAV2 capsid protein VP1, resulting in a DARPin-targeted AAV we termed DART-AAV. Stocks of mCD8-AAV contained similar genome copies as AAV2 but were >20-fold more active in gene delivery in mouse splenocytes, while exhibiting >99% specificity for CD8+ cells. These results suggest that receptor targeting can overcome blocks in transduction of mouse splenocytes.

In Vivo Generation of CAR T Cells Selectively in Human CD4+ Lymphocytes.

T cells modified with CD19-specific chimeric antigen receptors (CARs) result in significant clinical benefit for leukemia patients but constitute a challenge for manufacturing. We have recently demonstrated the in vivo generation of CD19-CAR T cells using the CD8-targeted lentiviral vector (CD8-LV). In this study, we investigated the in vivo generation of CD4+ CAR T cells using CD4-targeted LV (CD4-LV). Administration of CD4-LV into NSG mice transplanted with human peripheral blood mononuclear cells (PBMCs) led to 40%-60% of human CD4+ lymphocytes being CAR positive while CD8+ cells remained CAR negative. CAR+ T cells displayed a T helper 1 (Th1)/Th2 phenotype, which was accompanied by CD19+ B cell elimination. Intravenous administration of CD4-LV into NSG mice reconstituted with human CD34+ cells induced CAR expression and B cell elimination within 2-3 weeks post-injection. Preclinical analysis in a tumor mouse model revealed that mice administered CD4-LV exhibited faster and superior tumor cell killing compared to mice injected with CD8-LV alone or as a mixture with CD4-LV. Further analysis suggests that CD4+CAR+ cells may outperform CD8+CAR+ cells, especially at a high burden of target antigen, mainly since CD8 cells are more prone to exhaustion. This first description of in vivo-generated CD4+ CAR T cells supports their importance for cellular therapy.

CD8-Specific Designed Ankyrin Repeat Proteins Improve Selective Gene Delivery into Human and Primate T Lymphocytes.

Adoptive T cell immunotherapy in combination with gene therapy is a promising treatment concept for chronic infections and cancer. Recently, receptor-targeted lentiviral vectors (LVs) were shown to enable selective gene transfer into particular types of lymphocytes both in vitro and in vivo. This approach might facilitate the genetic engineering of a patient's own T lymphocytes, possibly even shifting this concept from personalized medicine to an off-the shelf therapy in future. Here, we describe novel high-affinity binders for CD8 consisting of designed ankyrin repeat proteins (DARPins), which were selected to bind to the CD8 receptor of human and nonhuman primate (NHP) cells. These binders were identified by ribosome display screening of DARPin libraries using recombinant human CD8 followed by receptor binding analysis on primary lymphocytes. CD8-targeted LVs (CD8-LVs) were then generated that delivered genes exclusively and specifically to human and NHP T lymphocytes by using the same targeting domain. These CD8-LVs were as specific for human T lymphocytes as their single-chain variable fragment-based counterpart, but they could be produced to higher titers. Moreover, they were superior in transducing cytotoxic T cells both in vitro and in vivo when equal particle numbers were applied. Since the here described CD8-LVs transduced primary T lymphocytes from NHP and human donors equally well, they offer the opportunity for preclinical studies in different animal models including large animals such as NHPs without the need for modifications in vector design.

Surface-Engineered Lentiviral Vectors for Selective Gene Transfer into Subtypes of Lymphocytes.

Lymphocytes have always been among the prime targets in gene therapy, even more so since chimeric antigen receptor (CAR) T cells have reached the clinic. However, other gene therapeutic approaches hold great promise as well. The first part of this review provides an overview of current strategies in lymphocyte gene therapy. The second part highlights the importance of precise gene delivery into B and T cells as well as distinct subtypes of lymphocytes. This can be achieved with lentiviral vectors (LVs) pseudotyped with engineered glycoproteins recognizing lymphocyte surface markers as entry receptors. Different strategies for envelope glycoprotein engineering and selection of the targeting ligand are discussed. With a CD8-targeted LV that was recently used to achieve proof of principle for the in vivo reprogramming of CAR T cells, these vectors are becoming a key tool to genetically engineer lymphocytes directly in vivo.

In vivo generation of human CD19-CAR T cells results in B-cell depletion and signs of cytokine release syndrome.

Chimeric antigen receptor (CAR) T cells brought substantial benefit to patients with B-cell malignancies. Notwithstanding, CAR T-cell manufacturing requires complex procedures impeding the broad supply chain. Here, we provide evidence that human CD19-CAR T cells can be generated directly in vivo using the lentiviral vector CD8-LV specifically targeting human CD8+ cells. Administration into mice xenografted with Raji lymphoma cells and human peripheral blood mononuclear cells led to CAR expression solely in CD8+ T cells and efficacious elimination of CD19+ B cells. Further, upon injection of CD8-LV into mice transplanted with human CD34+ cells, induction of CAR T cells and CD19+ B-cell depletion was observed in 7 out of 10 treated animals. Notably, three mice showed elevated levels of human cytokines in plasma. Tissue-invading CAR T cells and complete elimination of the B-lymphocyte-rich zones in spleen were indicative of a cytokine release syndrome. Our data demonstrate the feasibility of in vivo reprogramming of human CD8+ CAR T cells active against CD19+ cells, yet with similar adverse effects currently notorious in the clinical practice.